A novel ultrasound-guided mouse model of sudden cardiac …Jul 27, 2020 · 60 a rate of 150 bpm (125 µL for females and 140 µL for males). Animal temperature was maintained near

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1 A novel ultrasound-guided mouse model of sudden cardiac arrest

2 Cody A. Rutledge1, Takuto Chiba2,3, Kevin Redding1, Cameron Dezfulian4, Sunder Sims-Lucas2,3, Brett A. 3 Kaufman1

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5 1. Division of Cardiology, Cardiovascular Institute, University of Pittsburgh, Pittsburgh, PA, USA

6 2. Rangos Research Center, Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA, 7 USA

8 3. Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine, 9 Pittsburgh, PA, USA.

10 4. Safar Center for Resuscitation Research and Critical Care Medicine Department, University of 11 Pittsburgh, Pittsburgh, PA, USA

12

13 Corresponding Author:

14 Brett Kaufman ([email protected])

15

.CC-BY 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted July 27, 2020. ; https://doi.org/10.1101/2020.07.27.222695doi: bioRxiv preprint

https://doi.org/10.1101/2020.07.27.222695

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16 Abstract

17 Aim: Mouse models of sudden cardiac arrest are limited by challenges with surgical technique and 18 reliable venous access. To overcome this limitation, we sought to develop a simplified method in the 19 mouse that uses ultrasound-guided injection of potassium chloride directly into the heart.

20 Methods: Potassium chloride was delivered directly into the left ventricular cavity under ultrasound 21 guidance in intubated mice, resulting in immediate asystole. Mice were resuscitated with injection of 22 epinephrine and manual chest compressions and evaluated for survival, body temperature, cardiac 23 function, kidney damage, and diffuse tissue injury.

24 Results: The direct injection sudden cardiac arrest model causes rapid asystole with high surgical survival 25 rates and low surgical duration. Sudden cardiac arrest mice with 8-min of asystole have significant 26 cardiac dysfunction at 24 hours and high lethality within the first seven days, where after cardiac 27 function begins to improve. Sudden cardiac arrest mice have secondary organ damage, including 28 significant kidney injury, but no clear evidence of neurologic dysfunction.

29 Conclusions: Ultrasound-guided direct injection of potassium chloride allows for rapid and reliable 30 cardiac arrest in the mouse that mirrors human pathology. This technique lowers the barriers to entry 31 for adoption of the mouse model of sudden cardiac arrest, which will improve investigators’ ability to 32 study the mechanisms underlying post-arrest changes.



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33 Introduction

34 Out of hospital cardiac arrest affects over 350,000 patients annually in the United States1. Only 10% of 35 these patients will survive to hospital discharge and only 6% of the patients will be discharged with a 36 favorable outcome2,3. These statistics suggest a dramatic need for novel interventions to improve 37 cardiac arrest outcomes. Numerous animal models of sudden cardiac arrest (SCA) have been developed 38 to help understand the mechanisms underlying cardiac arrest mortality and to explore potential 39 interventions in pre-clinical models4,5. A review of animal models of SCA found that only 6% of pre-40 clinical SCA studies were completed in mice5. A mouse model has a number of advantages over large 41 animal models, including rapid breeding, cost-effectiveness, and opportunity for genetic manipulation.6 42 However, mouse use to model SCA has been limited by difficulty obtaining venous access and delivering 43 life support related to the animal’s size7,8.

44 The major limitation to adoption of the mouse model is the difficult cannulation of the delicate femoral 45 or internal jugular (IJ) veins, resulting in prolonged surgical times and high lethality. Moreover, venous 46 cannulation requires ligation of the vessel post-operatively, which may further contribute to organ 47 damage and alter outcomes. In this paper, we describe a novel method for ultrasound-delivery of 48 potassium chloride (KCl) directly into the left ventricle (LV) to induce immediate cardiac arrest, 49 bypassing the need for establishing intravenous access. This direct cardiac injection method simplifies 50 the surgical process, lowering the barrier to entry for this model. Our model has rapid procedure times 51 and high rate of surgical survival, thereby increasing the opportunity for study of preventative and 52 therapeutic interventions, as well as mechanistic aspects of organ damage and recovery.

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55 Methods

56 Animal Preparation

57 8-week-old C57BL/6J male and female mice were anesthetized using 5% isoflurane in 100% oxygen via 58 induction box until reaching the surgical plane. These mice were placed in a supine position and 59 intubated by 22 g catheter and mechanically ventilated (MiniVent, Harvard Apparatus, Holliston, MA) at 60 a rate of 150 bpm (125 µL for females and 140 µL for males). Animal temperature was maintained near 61 37 °C via heating pad and rectal temperature probe and heart rate (HR) was monitored continuously 62 using surface electrocardiogram (ECG; Visual Sonics, Toronto, Canada). HR was maintained between 63 400-500 bpm by adjusting isoflurane concentration. Depilatory cream was applied to the thorax and the 64 chest cleaned with alcohol. All studies were performed at the University of Pittsburgh in compliance 65 with the National Institutes of Health Guidance for Care and Use of Experimental Animals and was 66 approved by the University of Pittsburgh Animal Care and Use Committee (Protocol #18032212).

67 Ultrasound-guided KCl Delivery and Cardiopulmonary Resuscitation (CPR).

68 Baseline transthoracic echocardiography was performed using the Vevo 3100 imaging systems (Visual 69 Sonics) with a 40 MHz linear probe along the long-axis of the heart. A 30-gauge needle was carefully 70 advanced under ultrasound-guidance through the intercostal space and directed into the LV (Figure 1A). 71 40 µL of 0.5M KCl in saline was delivered into the LV cavity, causing immediate asystole on ECG. The 72 ventilator was turned off at this time. Doppler imaging was utilized over the aortic outflow tract to 73 confirm that no blood was ejected during asystole. The mice remained in asystole for 7.5 minutes, when 74 a second 30-gauge needle was introduced into the LV and 500 µL of 15 µg/mL epinephrine in saline was 75 delivered over approximately 30 seconds. At 8 minutes, the ventilation was resumed at 180 bpm and 76 CPR initiated. CPR was performed manually at 300 bpm for 1 min, at which time CPR was briefly held 77 and ECG evaluated for recovery of spontaneous circulation (ROSC). If a ventricular rhythm was observed 78 on ECG, doppler imaging was performed to confirm blood flow. If not, one to two additional 1-minute 79 cycles of CPR were performed. Animals not achieving ROSC by 3 minutes were euthanized. Mice 80 remained on the ventilator (without isoflurane) for approximately 20-25 minutes until breathing 81 spontaneously at a rate over 60 respirations/minute. Sham mice received no KCl injection, but did 82 receive a single direct LV injection of 500 µL epinephrine and were extubated immediately after 83 injection. All animals were placed in a recovery cage under heat lamp for 2 hours with hourly 84 temperature monitoring for up to 4 hours. Survival was assessed every morning for a 4-week survival 85 cohort.

86 Figure 1. Direct LV Injection Model of SCA. A) Representative long-axis ultrasound image depicting 87 introduction of a needle into the LV chamber. B) Representative ECG tracings at baseline, during KCl 88 injection, during asystole, immediately after ROSC is achieved, and during recovery. C) Depiction of time 89 course of SCA in this model. D) Temperature monitoring in sham and arrest mice at baseline, time of 90 ROSC, and at 1, 2, 3, 4 and 24 hours post ROSC. Arrest mice have significanly depressed body 91 temperature at 3, 4, and 24 hours after arrest when compared to sham mice. E) Mortality curve 92 demonstrating descreased survival in arrest mice as compared to sham (initial sham n=5; arrest n=13). 93 Data are expressed as mean +/- SEM. *=p < 0.05

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95 Ultrasound and Echocardiography

96 Echocardiography was performed at baseline, 1 day, 1 week, and 4 weeks as previously described14. 97 Briefly, transthoracic echocardiography was performed using the Vevo 3100 system and analyzed using 98 VevoLab v3.2.5 (Visual Sonics). B-mode images were taken for at least 10 cardiac cycles along the 99 parasternal long axis of the LV and end-systolic volume (ESV) and end-diastolic volumes (EDV) calculated

100 by modified Simpson’s monoplane method15. Short-axis M-mode images were obtained at the level of 101 the papillary muscle for representative images only. Ejection Fraction (EF) was calculated from long-axis 102 B-mode imaging as: 100 × (LV EDV − LV ESV) / (LV EDV).

103 A cohort of mice were assessed for renal perfusion following ROSC. The ultrasound probe was oriented 104 transversely across the abdomen at the plane of the right kidney. Doppler imaging over the renal artery 105 evaluated the presence of blood flow every thirty seconds until sustained blood flow was noted.

106 Serum Analysis

107 After euthanasia, mice underwent cardiac puncture for collection of blood by heparinized syringe. Blood 108 was separated by centrifugation at 2000 x g at 4 °C for 10 minutes and the serum was flash frozen. 109 These samples were evaluated for blood urea nitrogen (BUN), serum creatinine, alanine 110 aminotransferase (ALT), and creatine kinase (CK) by the Kansas State Veterinary Diagnostic Laboratories 111 (Manhattan, KS).

112 Tissue Histology

113 Kidneys were fixed overnight in 10% formalin at 4 °C then washed with PBS and transferred to 70% 114 ethanol at room temperature. After fixation, tissues were embedded into paraffin prior to sectioning at 115 4 microns by the Histology Core at the Children’s Hospital of Pittsburgh. Sections were stained with 116 hematoxylin and eosin (H&E). Renal tubular pathology was semi-quantitatively scored (0: no injury to 4: 117 severe injury) in terms of tubular dilatation, formation of proteinaceous casts, and loss of brush 118 border16. Histological scoring was performed in a blinded fashion at 40x magnification on outer 119 medullary regions of the tissue sections. Eight fields were evaluated per sample. Samples were imaged 120 using a Leica DM 2500 microscope (Leica, Wetzlar, Germany) and LAS X software (Leica).

121 Statistical Analysis

122 Data were expressed and mean ± standard error in all figures. p ≤ 0.05 was considered significant for all 123 comparisons. To determine whether sample data has been drawn from a normally distributed 124 population, D’Agostino-Pearson test was performed. For parametric data, Student’s t-test was used to 125 compare two different groups. For nonparametric data, Mann-Whitney test was used. Survival analysis 126 was assessed by using Kaplan-Meier and log rank (Mantel-Cox) testing. All statistical analysis was 127 completed using Graphpad Prism 8 software (San Diego, CA).

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129 Results

130 Baseline sex, weight, EF and HR are similar between groups

131 19 sham and 30 arrest mice were initially utilized in this study. There was no difference in baseline 132 weight (sham: 22.6±0.9 g; arrest: 23.1±0.6 g, Table 1), EF (sham: 59.8±1.5%; arrest: 59.9±1.0%) or HR 133 (sham: 472±19 bpm; arrest: 488±11 bpm) between groups. A single mouse from the sham group (1/19, 134 5.2%) and five mice from the arrest group (5/30, 16.7%) did not achieve ROSC or died immediately after 135 extubation. Arrest mice required an average of 1.32 minutes of CPR to achieve ROSC and were 136 extubated after an average of 22.7 minutes (Table 1). The distribution of males and females are similar 137 between groups (sham: 10 female, 9 male; arrest: 14 female, 16 male). While this study was not 138 powered to examine sex-based changes amongst groups, surgical survival was not biased by sex 139 distribution (Table 1).

140 Table 1. Physiologic and Surgical Characteristics of Sham and SCA Mice

Sham (±SEM) Arrest p-value

Age (d) 56.8±0.7 (n=18) 57.7±0.6 (n=25) 0.34

Weight (g) 22.6±0.9 (n=15) 23.1±0.6 (n=25) 0.63

Sex 10 female, 9 male 14 female, 16 male n/a

Surgical Survival

Total

Males

Females

18/19

9/9

9/10

25/30

14/16

11/14

n/a

CPR Duration n/a 1.32±0.11 (n=25) n/a

Time to Extubation n/a 22.7±0.7 (n=25) n/a

Initial Body Temp (°C) 35.7±0.2 (n=15) 35.5±0.2 (n=25) 0.79

ROSC Body Temp (°C) n/a 35.2±0.2 (n=25) n/a

1 h Body Temp (°C) 35.9±0.1 (n=7) 35.9±0.3 (n=14) 0.95

2 h Body Temp (°C) 35.8±0.1 (n=7) 35.2±0.3 (n=13) 0.18

3 h Body Temp (°C) 35.8±0.1 (n=6) 33.0±0.6 (n=9) <0.001

4 h Body Temp (°C) 35.8±0.1 (n=6) 32.9±0.1 (n=5) <0.001

24 h Body Temp (°C) 36.6±0.2 (n=7) 34.8±0.4 (n=10) 0.03

Baseline HR (bpm) 472±19 (n=13) 488±11 (n=19) 0.45

Baseline EF (%) 59.8±1.5 (n=14) 59.9±1.0 (n=19) 0.94



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1 d HR (bpm) 521±18 (n=14) 459±12 (n=21) 0.007

1 d EF (%) 59.6±1.7 (n=14) 39.9±3.0 (n=21) <0.001

1 wk EF (%) 59.6±2.3 (n=5) 41.4±3.4 (n=6) 0.002

4 wk EF (%) 59.5±2.6 (n=5) 49.8±5.3 (n=6) 0.4

141

142 SCA mice have temperature and HR dysregulation after ROSC

143 There were no significant differences in body temperature between groups at baseline (sham: 35.7±0.2 144 °C; arrest: 35.5±0.2 °C) (Table 1, Figure 1). Following extubation, mice were kept in a warmed recovery 145 cage, and no difference was noted at 1 hour (sham: 35.9±0.1 °C; arrest: 35.9±0.3 °C) or 2 hours (sham: 146 35.8±0.1 °C; arrest: 35.2±0.3 °C). Arrest mice had significantly lower body temperatures once removed 147 from the warming cage at 3 hours (sham: 35.8±0.1 °C; arrest: 33.0±0.6 °C, p<0.001), 4 hours (sham: 148 35.8±0.1 °C; arrest: 32.9±0.1 °C, p<0.001) and 24 hours (sham: 36.6±0.2 °C; arrest: 34.8±0.4 °C, p=0.03). 149 Arrest mice also had significantly lower HR one-day after SCA (sham 521±18 bpm; arrest 459±12 bpm, 150 p=0.007).

151 SCA mice have increased 30-day mortality

152 Of the post-operative survivors, a cohort from each group was evaluated for survival over a 4-week time 153 course. At 24 hours, 100% of sham mice survived (5 of 5) compared to 92% of arrest mice (12 of 13, 154 p=0.54). At 72 hours, 100% of sham mice were alive (5 of 5) compared to 46% of arrest mice (6 of 13, 155 p=0.052 vs sham). At 4 weeks, 100% of sham mice survived (5 of 5, median survival 28 days) compared 156 to only 38% of arrest mice (5 of 13, median survival 3 days, p=0.03; Figure 1). All of the SCA mouse 157 deaths occurred within seven days following arrest.

158 SCA mice have reduced EF, which improves over time

159 Sham and arrest mice showed no difference in baseline EF (sham: 59.8±1.5%; arrest: 59.9±1.0%). One 160 day after arrest, there was a significantly depressed EF in the arrest group (sham: 59.6±1.7 %; arrest: 161 39.9±3.0%, p<0.001; Figure 2). EF of arrest mice remained significantly depressed 1 week after SCA 162 procedure (sham: 59.6±2.3%; arrest: 41.4±3.4%, p=0.002). Four weeks after arrest, there is no 163 significance between EF of sham and SCA groups (Figure 2).

164 Figure 2. Ejection Fraction and Heart Rate in Sham and SCA Mice. A) Representative M-mode tracings 165 of a sham (top) and arrest (bottom) mouse one day after SCA, where red lines denote LV width during 166 diastole and green lines denote systole. B) Heart rate (HR) and ejection fraction (EF) are similar between 167 groups at baseline, but significantly depressed in SCA mice compared to sham at 1 day after arrest. C) EF 168 is significantly decreased in SCA mice compared to sham at matched timepoints at 1 day and 1 week, but 169 there is no significant EF change by 4 weeks. Data are expressed as mean +/- SEM. P-values: *< 0.05, ** 170 < 0.01, ***< 0.001.

171 SCA mice have evidence of prolonged ischemia after SCA and kidney damage at one day

172 As kidney damage is a common side effect of cardiac injury17, we evaluated the duration of renal 173 ischemia following SCA. A cohort of arrest mice (n=10) were evaluated for kidney reperfusion following



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174 ROSC by evaluating renal artery flow. The mean duration of kidney ischemia was 20.6 minutes, with 175 initial measurable kidney blood flow occurring on average 11.3 minutes after ROSC (Figure 3). Serum 176 creatinine was significantly elevated in arrest mice at 1 day when compared to sham (sham: 0.36±0.06 177 mg/dL; arrest: 1.52±0.22 mg/dL, p<0.001; Figure 3), as was serum BUN (sham: 21.5±9.9 mg/dL; arrest: 178 156.0±39.8 mg/dL, p=0.005). Semi-quantitative scoring of tubular injury was performed at the outer 179 medulla and was noted to be higher in arrest mice (sham: 0.15±0.05; arrest: 3.33±0.29, p<0.0001).

180 Figure 3. Kidney, Neurologic, and Serum Chemistry 1-day after SCA. A) Percentage of mice with 181 recovered kidney perfusion over time since arrest (n=10). Mean recovery time was 20.55±0.68 min. B) 182 SCA mice have increased kidney damage by semi-quantitative scoring of kidney injury in the outer 183 medulla (n=5/group). C) Representative H&E stains of sham (top) and arrest (bottom) mice one-day after 184 arrest demonstrating proteinaceous casts in renal tubules (black arrowhead) and infiltrates (red 185 arrowheads) with glomeruli marked (blue asterisk). D) Elevated serum creatinine and BUN in SCA mice at 186 one-day. E) Neurologic scoring at one day. F) ALT, CK, and lactate changes at one day. Data are 187 expressed as mean +/- SEM. P-values: *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001.

188 SCA mice have diffuse tissue injury at one day

189 To assess systemic damage, additional serum assays were performed at 1 day to evaluate for liver 190 damage (ALT), muscle damage (CK), tissue ischemia (lactate), and neurologic function. These assays 191 were notable for a significant increase in ALT (sham: 47.6±4.7 U/L; arrest: 135.6±37.3 U/L, p=0.047; 192 Figure 3) in arrest mice compared to sham. No significant changes were noted to serum CK (sham: 193 1244±252 U/L; arrest: 1811±570 U/L, p=0.36) or normalized serum lactate (sham: 1.00±0.06; arrest: 194 1.90±0.64, p=0.15). Brief neurologic testing was performed as previously reported11 on 14 sham and 17 195 arrest mice one day after SCA (Table 2). Two of the arrest mice were noted to have hind-leg ataxia and 196 one mouse had sluggish movement; however, there was no significant difference in neurologic testing 197 between the groups (sham: score 12.0±0; arrest: score 11.8±0.1, p=0.13; Figure 3).

198 Table 2. 12-point Neurologic Function Assessment for One-Day Sham and SCA Mice

Neurological Function

Level of Consciousness

No Tail Pinch Reflex 0

Weak Tail Pinch Reflex 1

Normal Tail Pinch Reflex 2

Corneal Reflex

No Blink 0

Delayed Blink 1

Normal Blink 2

Respiration

Irregular 0



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Decreased Frequency with Normal Pattern 1

Normal Frequency and Pattern 2

Righting Reflex

No Righting 0

Sluggish Righting 1

Rapid Righting 2

Coordination

No Movement 0

Some Ataxia 1

Normal Coordination 2

Activity

No Spontaneous Movement 0

Sluggish Movement 1

Normal Movement 2

Total Possible Score 12

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200 Discussion

201 In this study, we modified the mouse model of SCA described by Hutchens et al.9 by delivering KCl 202 directly into the LV cavity under ultrasound guidance rather than IJ cannulation. This delivery method 203 causes immediate onset of asystole after KCl delivery in a highly controlled, easily visualized, and easily 204 adoptable manner. Pre-clinical models of SCA are rarely performed in the mouse despite a large number 205 of advantages of the murine model, including rapid development, low maintenance cost, and the 206 opportunity for genetic manipulation6,7. The low utilization of the murine model is likely attributable to 207 surgical difficulties related to animal size. Groups that have embraced the mouse model of SCA almost 208 uniformly rely on intravenous delivery of KCl for induction of cardiac arrest, but have utilized various 209 durations of arrest (typically 4-16 minutes). These reports all use venous access either through the 210 jugular or femoral veins for drug delivery. Establishing reliable venous access in the mouse remains a 211 major barrier to wide-spread adoption of the mouse model of SCA.

212 Our model spares the use of major vessels for drug delivery by using direct LV introduction under 213 ultrasound, resulting in rapid procedure times, typically around 30 minutes from anesthesia induction to 214 extubation, with low surgical mortality (16.7% of SCA mouse surgical mortality, Table 1). Ultrasound-215 guided catheter placement has already become a staple of hospital care for many clinicians, allowing 216 physicians and researchers to easily transfer a known skill set into a translational model of SCA18,19. 217 Additionally, the destruction of major veins by cannulation in the traditional mouse model may limit



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218 venous drainage post-operatively, which may affect both neurologic and cardiac performance. Our 219 method avoids both venous access and blockage. Further, ultrasound utilization allows for continual 220 non-invasive monitoring of cardiac function throughout the arrest, resulting in precise monitoring of 221 cardiac arrest duration. Previously published models that utilize only ECG as an indicator of ROSC may 222 falsely register pulseless electrical activity as a return of circulation, which may erroneously measure 223 ischemic duration. Other models utilize an LV pressure catheter to accurately record restoration of 224 cardiac flow; however, this requires the placement of an additional invasive canula. In the current 225 report, the rapid procedure time, high survival, reduced surgical skill required, and venous sparing by 226 this technique are highly advantageous over the traditional model.

227 Of the 30 mice that underwent arrest in this study, 25 survived the arrest and achieved ROSC, resulting 228 in a relatively low mortality rate (16.7%) for the procedure (Table 1). This is a modest improvement over 229 the 20% surgical mortality in the venous KCl mouse model described by Hutchens et al.9 and the 27% 230 mortality in a ventricular fibrillation mouse model described by Chen et al.8 A subset of mice was studied 231 for up to 4 weeks to assess long-term survival. At 72 hours, 6 of 13 arrest mice (46%) survived, which is 232 in line with comparable recent studies publishing between 10 and 45% survival at 72 hours20–22. 5 of 13 233 mice (38%) survived for the entire duration of the study (Figure 1).

234 After one day, EF in SCA mice is significantly decreased from 59.9% at baseline to 39.9%. These values 235 are in-line with previously published one day EFs and are likely attributable to cardiac stunning21. EF 236 remains significantly decreased at one week (41.4%), with improvement at four weeks (49.8%, Figure 2). 237 These values and their relative improvement are similar to those observed in humans following cardiac 238 arrest in the absence of coronary disease, as evidenced by a case series of cardiac arrest survivors, which 239 noted 1 day EFs of 38%, 1 week EFs of 44%, and 2-3 week EFs of 50%23.

240 Maintenance of body temperature is critically important to neurologic outcomes following SCA24–26. We 241 maintained body temperatures with active heating for 2 hours after arrest, but body temperatures fell 242 significantly after active heating was stopped, which is consistent with post-arrest changes in humans27. 243 The SCA mice continued to show significant temperature dysregulation and depressed HR at one day 244 (Figure 1, Table 1). We were unable to demonstrate significant neurologic deficit 24 hours after arrest by 245 utilizing a well-established, 12-point examination11. Delayed, spontaneous hypothermia is known to be 246 neuroprotective in other rodent cardiac arrest models and may explain the paucity of neurological injury 247 noted28. We only noted ataxia in two mice and lethargy in one mouse following SCA, with no observable 248 deficits in sham mice. While some groups are able to demonstrate neurologic injury with as little as 6-249 minutes of cardiac arrest29, others have required extended arrest time of 12-14 minutes to detect 250 neurologic changes10,30. The technique described in this paper would be easily modified to allow for 251 prolonged arrest time to study neurologic insult.

252 Mouse SCA models have been utilized to as a clinically-relevant model of both acute kidney injury31,32 253 (one day after arrest) and chronic kidney disease (seven weeks after arrest, attributed to prolonged 254 inflammation after reperfusion)32. We show that our model of SCA similarly develops markers of AKI, as 255 evidenced by elevated serum creatinine, BUN, and tubular damage 1 day after arrest (Figure 3). Many of 256 the peripheral kidney tubules are anucleated one-day after arrest, which is a marker of nephrotic 257 damage in mice. Kidney injury is not typically apparent with 8 minutes of direct ischemia, which typically 258 require 15-20 minutes for the development of focal injury33. By utilizing doppler ultrasonography of the 259 renal artery, we found that the kidney did not receive measurable perfusion until 11.25 minutes after



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260 ROSC, for a total mean ischemia duration of 20.55 minutes, resulting in kidney ischemia times consistent 261 with established direct ischemia reperfusion injury models (Figure 3)34,35.

262 Finally, our model suggests the presence of global ischemic injury following SCA. ALT, a non-specific 263 marker of liver injury, is significantly elevated one-day after SCA (Figure 3). However, there are non-264 significant trends towards increased CK, a non-specific marker of muscle degradation, and serum lactate 265 levels in the SCA mice. These changes are consistent with the markers of secondary ischemic damage 266 seen in post-cardiac arrest patients. We anticipate that these changes could become significant with 267 prolonged arrest duration, but at the cost of increased mortality. Further evaluation of this multiple 268 organ damage and the degree to which each organ system is involved may be an important step toward 269 improving recovery and guiding post-cardiac arrest interventions.

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271 Conclusions

272 We demonstrate a novel mouse model of SCA that utilizes direct LV injection of KCl under ultrasound 273 guidance that allows for rapid and reliable arrest with low surgical mortality. This model develops 274 significant cardiac, kidney, and liver injury at one day and a trend towards neurologic injury. Time of 275 injury but can be easily adapted to achieve neurologic insult. This model lowers the barrier to entry for 276 establishing a mouse model of SCA, which will help researchers investigate the mechanisms underlying 277 SCA mortality.

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A novel ultrasound-guided mouse model of sudden cardiac …Jul 27, 2020 · 60 a rate of 150 bpm (125 µL for females and 140 µL for males). Animal temperature was maintained near